Most conventional pharmaceuticals currently in use for the treatment of serious disorders such as cancer and inflammatory diseases do not selectively accumulate at the site of disease [Bosslet et al., 58, 1195-1201 Cancer Res. (1998)]. For example, intravenously administered drugs distribute evenly within the different organs and tissues of the body, rather than selectively accumulating at the site of disease.
One approach to circumvent the disadvantages of conventional pharmacological therapies involves the preferential delivery of a bioactive agent to the site of disease by means of a binding molecule specific for a pathology-associated marker [Neri & Bicknell (2005) Nature Rev. Cancer, 4, 436-446]. The selective targeting of the drug to the diseased tissue will ultimately result in an increased local concentration at its site of action, sparing normal organs from the unwanted effects of the bioactive agent used to confer a pharmacological benefit (e.g., a growth factor, an enzyme, a hormone, an anti-inflammatory drug, a cytotoxic drug, a cytokine, a radionuclide, or a photosensitizer). In most cases, this will lead to an improved therapeutic index of the delivered pharmaceutical, i.e. a higher efficacy with minimized side effects. Indeed, the favourable toxicity profile of site-specific therapeutics may open new avenues in the therapy of angiogenesis-related diseases, allowing the systemic administration of highly potent and promising agents, which are currently either given at suboptimal doses or whose clinical application has to date been impeded by unacceptable side-effects when applied in an unmodified form.
Ligand-based pharmacodelivery strategies fundamentally rely on the identification of good-quality markers of pathology, allowing a clear-cut discrimination between diseased tissues and healthy organs. Monoclonal antibodies and their fragments represent the preferred agents for pharmacodelivery applications [Rybak et al. 2, 22-40 Chem. Med. Chem (2007); Shrama et al., 5, 147-159 Nat. Rev. Drug Discovery (2006)], but globular protein mutants [Binz and Plückthun, 23, 1257-1268 Nature Biotechnology (2005)], peptides [Sergeeva et al., 58, 1622-1654, Adv. Drug. Deliv. Rev. (2006)] and even small organic ligands [Low et al., 41, 120-129, Acc. Chem. Res. (2008)] are also increasingly being used.
Antibody-based targeted delivery of bioactive agents to sites of angiogenesis as a therapeutic strategy for cancer treatment has been described. In the case of inflammatory disorders, antibody-based targeted delivery is much less well studied. The applicant has previously demonstrated that the ED-A domain of fibronectin, is expressed in Inflammatory Bowel Disease. Using both radioactive and fluorescent techniques, the human monoclonal antibody F8, specific to ED-A, was found to selectively localize at sites of inflammation in vivo, following intravenous administration (WO2014/055073).
However, there remains a need in the art for further antibodies which can be employed in ligand-based pharmacodelivery applications for the treatment and diagnosis of inflammatory disorders and autoimmune diseases, such as IBD.
Lysozyme
Lysozyme, is a glycoside hydrolase enzyme that damages bacterial cell walls by catalysing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins. Lysozyme is known to be expressed by the epithelial cells of in the mucosa of active IBD [Klockars et al., 18, 377-341, Gut (1977), Montero et al., 190, 127-142, Anat Rec. (1978), Cunliffe et al., 55, 298-304, J Clin Pathol (2002) Rubio C A, 3,73-92, Pathogens (2014)].
Neutrophil Elastase
Neutrophil elastase is a serine proteinase secreted by neutrophils and macrophages during inflammation. As with other serine proteinases it contains a charge relay system composed of the catalytic triad of histidine, aspartate and serine residues that are dispersed throughout the primary sequence of the polypeptide but that are brought together in the three dimension conformation of the folded protein.
It has been reported that neutrophil elastase activity is elevated in both colonic mucosa and blood in inflammatory bowel disease (IBD) patients, and that it can act as an aggravating factor in IBD. [Gouni-Berthold et al., 46, 2315-2320, Hepatogastroenterol (1999), Shioya et al., 60, 14-21 Fukushima J Med Sci (2014)]. It is also a marker capable of differentiating active inflammatory bowel disease from inactive inflammatory bowel disease and irritable bowel syndrome (IBS) [Langhorst et al., 103, 162-169, Am J Gastroenterol (2008)].
Tissue Inhibitor of Metalloproteinase-1 (TIMP-1)
Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a glycoprotein that inhibits the matrix metalloproteinases (MMPs), a group of peptidases involved in the degradation of the extracellular matrix. In addition to its inhibitory role against most of the known MMPs, the encoded protein is able to promote cell proliferation in a wide range of cell types, and may also have an anti-apoptotic function.
Serum concentrations of TIMP-1 have been shown to be significantly increased in patients with ulcerative colitis and Crohn's Disease compared with controls [Lacatos et al., 30, 289-295, Dig Dis (2012)].
IIICS Isoform of Fibronectin
Fibronectins (FN) are multifunctional, high molecular weight glycoprotein constituents of both the extracellular matrix and body fluids. They are involved in many different biological processes such as the establishment and maintenance of normal cell morphology, cell migration, haemostasis and thrombosis, wound healing and oncogenic transformation [Alitalo et al., (1982) Adv Cancer Res, 37 111-158; Yamada, (1983) Curr Opin Cell Biol, 1, 956-963; Hynes, (1985) Annu Rev Cell Biol, 1, 67-90; Ruoslahti et al., (1988) Annu Rev Biochem, 57, 375-413; Owens et al., (1986) Oxf Sury Eukaryot Gene, 3, 141-160]. Structural diversity in FNs is brought about by alternative splicing of three regions (ED-A, ED-B and IIICS) of the primary FN transcript (Hynes, R., (1985) Annu Rev Cell Biol, 1, 67-90; Zardi et al., (1987) EMBO J, 6, 2337-2342) to generate at least 20 different isoforms, some of which are differentially expressed in tumour and normal tissue. For example, five different splice isoforms of the human IIICS isoform of fibronectin have been described. As well as being regulated in a tissue- and developmentally specific manner, it is known that the splicing pattern of FN-pre-mRNA is deregulated in transformed cells and in malignancies (Castellani et al., et al., (1986) J Cell Biol, 103, 1671-1677; Borsi et al., (1987) J Cell Biol, 104, 595-600; Vartio et al., (1987) J Cell Sci 88, 419-430, Zardi et al., (1987) EMBO J, 6, 2337-2342; Barone et al., (1989) EMBO J, 8, 1079-1085; Carnemolla et al., (1989) FEBS Letter 215, 269-273; Oyama et al., (1989) Biochemistry, 28, 1428-1433; Borsi et al., (1992) Exp Cell Res 199, 98-105). The FN isoforms containing the ED-A, ED-B and IIICS sequences have been shown to be expressed to a greater extent in transformed and malignant tumour cells than in normal cells.
Much of the information relating to the expression of the IIICS isoform of fibronectin in healthy and diseased tissues derives either from mRNA studies or from studies with monoclonal antibodies (antibodies FDC-6 and X18A4). These antibodies were generated by hybridoma technology following immunization with fibronectin and immunosuppression with cyclophosphamide. Antibody FDC-6 binds to a specific O-linked N-acetygalactosaminylated hexapeptide epitope within the fibronectin type III connecting segment (IIICS) [Matsuura et al., (1985) PNAS, 82, 6517-6521; Matsuura et al., (1988) J Biol Chem, 263, 3314-3322]. However, since the antibody requires both the peptide backbone and the carbohydrate moiety to recognize the epitope, it is not suitable for targeting application especially when cross-reactivity between species is needed. Antibody X18A4 recognizes a different IIICS region than FDC-6, but the binding epitope has never been fully characterized [Feinberg R. et al., (1995) Am J Obstet Gynecol, 172, 1526-1536]: the main application for antibody X18A4 is related to the detection of oncofetal fibronectin in the cervix of pregnant women to predict preterm labour. There is evidence that IIICS expression is modulated in rheumatoid arthritis and osteoarthritis: in particular, it seems that the isoform 89V (CS1) is up-regulated in inflammation [Kriegsmann J et al., (2004) Rheumatol Int, 24, 25-33; Elices M J et al., (1994) J Clin Invest, 93, 405-416]. However, to our knowledge there is no report on the presence or role of IIICS in Inflammatory Bowel Disease.
ED-B Domain of Fibronectin
Fibronectin is a large glycoprotein that is present in large amounts in plasma and tissues. ED-B is a 91-amino-acid type III homology domain that becomes inserted into the fibronectin molecule under tissue-remodeling conditions by a mechanism of alternative splicing at the level of the primary transcript. The ED-B sequence is identical in mouse, rat, rabbit, dog, monkey and man and ED-B is essentially undetectable in healthy adult individuals with the exception of some vessels in the ovaries and the endometrium during the proliferative phase, when physiological angiogenesis is occurring.
However, ED-B-containing fibronectin has been shown to be abundant in many aggressive solid tumours, and displays either predominantly vascular or diffuse stromal patterns of expression, depending on the tumour. The presence of ED-B has also been reported in ocular angiogenesis [Birchler M et al., (1999) Nature Biotech, 17, 984-988, Nicolò Met al (2003) Am J Ophtalmol, 135, 7-13], rheumatoid arthritis (WO2007/128563), endometriosis [Schwager C et al., (2011) Hum Reprod, 26, 2344-2352, WO2010/078950] and atherosclerotic plaques [Matter C M et al., (2004) Circ Res, 95, 1225-1233, Pedretti M et al (2010) Atherosclerosis, 208, 382-389]. The applicant of the present application, has previously shown that the ED-B of fibronectin scored negative when probed with the anti ED-B antibody L19, in specimens of ulcerative colitis (WO2010/078950)
Matrix-Metalloproteinase 3 (MMP3)
Matrix metalloproteinase 3 (also known as stromelysin 1) is a member of a family of more than 20 zinc-dependent extracellular enzymes with a key role in tissue remodeling [Nagase 30 and Woessner, (1999) J Biol Chem, 274, 21491-21494; Martin and Matrisian, (2007) Cancer Metastasis Rev, 26, 717-724; Vartak and Gemeinhart, (2007) J Drug Target 15(1) 1-20].
Abnormal expression of various MMP proteins has been shown to play a role in a variety of disease types including cancer progression and in inflammatory conditions such as rheumatoid arthritis [Martin and Matrisian, (2007) Cancer Metastasis Rev, 26, 717-724; Brinckerhoff and Matrisian, (2002) Nat Rev Mol Cell Biol, 3, 207-214; Overall and Kleifeld, (2006) Nat Rev Cancer, 6, 227-239]. In a Crohn's Disease genome micro-array it has been reported that the MMP3 gene is differentially expressed compared to controls [Noble et al., (2010) Inflamm Bowel Dis, 16, 1717-1728]
A1, C and D Domains of Tenascin-C
Tenascin-C is a glycoprotein of the extracellular matrix. It comprises several fibronectin type 3 homology repeats that can be either included or omitted in the primary transcript by alternative splicing, leading to small and large isoforms that have distinct biological functions. Whereas the small isoform is expressed in several tissues, the large isoform of tenascin-C exhibits a restricted pattern of expression. It is virtually undetectable in healthy adult tissues but is expressed during embryogenesis and is expressed in adult tissues undergoing tissue remodeling including neoplasia.
Traditionally, one has referred to the large isoform of tenascin-C for tenascin molecules, which would putatively comprise all alternatively spliced domains A1, A2, A3, A4, B, AD, C, D, and to the small isoform of tenascin-C whenever these domains were absent. There are several reports indicating the presence of tenascin-C in general, in the serum and in the colonic tissues of patients with IBD [Riedl et al., 16, 285-291, Int J Colorectal Dis (2001), Geboes et al., 9, 281-286, Int J Surg Pathol (2001), Dueck et al., 82, 477-483, Int J Cancer (1999)]. However, the role of the A1 and of the D domain of Tenascin-C has not been elucidated in full and is not clear whether they can be used as a target for the pharmacodelivery of agents to treat or diagnose IBD. For example, the applicant of the present application, has previously shown that the domain A1 of Tenascin-C scored negative when probed with the anti-Al domain antibody F16, in specimens of ulcerative colitis (WO2010/078950). Antibodies that bind to the domain D of Tenascin-C and in particular the properties of the P12 antibody for tumor targeting have been described (Brack et al., Clin. Cancer Res. (2006) 12, 3200-3208 and in WO2006/050834).